HIV-1 isolates Ug06 and Ug23 were established in culture from peripheral blood mononuclear cells (PBMCs) of Ugandan subjects. The isolates were studied for phylogenetic and serological relationships with each other and with the laboratory strains, HTLV-IIIB and HIV-1MN. The results suggest that the Ugandan isolates are related to different subgroups of African viruses with 17.3% of genetic distance between UG06 and the U455 provirus (Uganda); and 12.6% of genetic distance between UG23 and the JY1 provirus (Zaire). Analysis of the predicted amino acid sequences for Ug06 and Ug23 showed marked sequence heterogeneity in the V3 region and CD4-binding site. A conserved amino acid sequence was identified in the C-terminal immunodominant region of the envelope glycoprotein gp120. The isolates were compared in virus-neutralization experiments with HTLV-IIIB and HIV-1MN stocks, using panels of Western blot-positive North American and Ugandan sera. The North American serum samples showed broad neutralizing activity against both of the Ugandan isolates. However, the Ugandan serum panel demonstrated strain-specific activity against either Ug06 or Ug23. Furthermore, the African serum specimens showed higher prevalence and titers of neutralizing activity against the HIV-1MN stock as compared with HTLV-IIIB.